Disruption of Clever-1 for Novel Anticancer Immunotherapy

The progression of cancer is dependent on the quality of the immune response elicited by the tumour. Typically, proinflammatory immune responses restrain and anti-inflammatory immune responses promote the progression of cancer. Thee immunosuppression caused by anti-inflammatory leukocytes, such as regulatory T cells and alternatively activated macrophages, is a major obstacle in the successful treatment of cancer. Clever-1 is a multifunctional protein expressed by a subset of human monocytes and alternatively activated, or M2, macrophages. In these macrophages, Clever-1 is involved in receptor-mediated endocytosis, intracellular sorting, and transcytosis. Clever-1 is also expressed on lymphatic and vascular endothelia, where it mediates the trafficking of leukocytes from the lymph or blood into tissues. Lymphatics and macrophages expressing Clever-1 have been detected in human cancers, and high numbers of Clever-1-positive macrophages correlate with poorer prognoses in advanced colorectal cancer. By using specific antibodies to interfere with the function Clever-1, we have recently demonstrated that Clever-1 antibody treatment limits the progression of cancer in animal models of melanoma and lymphoma. According to these results, the antibody-mediated interference of Clever-1 both before and after cancer cell implantation limits tumour growth and metastasis. However, the molecular mechanisms by which Clever-1 regulates the phenotype and function of tumour-associated macrophages during this process are very poorly understood. The data presented in this master’s thesis reveal novel molecular mechanisms employed by Clever-1 to regulate the function of M2 macrophages and suggest that Clever-1 antibody treatment may have immunotherapeutic potential in the treatment of breast cancer. According to the obtained results, Clever-1 promoted intracellular signal transduction through the anti-inflammatory mTORC1 complex in M2 macrophages. In accordance, the antibody-mediated interference of Clever-1 induced a proinflammatory response from M2 macrophages by increasing the production and secretion of TNF-alpha, a major mediator of inflammation. Furthermore, combinatorial antibody-mediated interference of Clever-1 and the immune checkpoint protein PD-1 decreased the relative amount of anti-inflammatory tumour-associated macrophages, thereby likely promoting a more proinflammatory milieu within the tumour microenvironment. Additionally, combinatorial Clever-1 and PD-1 antibody treatment appeared to limit tumour growth and metastasis also in a mouse model of triple-negative breast cancer.Siirretty Doriast

Re-educating macrophages to activate antitumor immunity: One Clever Immunotherapy

The immune system is our constant protector against external foes but also against our own incipient malignant cells. Unfortunately, developing cancers often learn to shut down the antitumor immune response or even to manipulate the immune system to support their own growth and progression instead. Checkpoint blockers that reactivate adaptive antitumor immunity have revolutionized cancer treatment—however, benefiters are in the minority. Therefore, novel treatment options are required for patients whose cancers are refractory. Tumor-associated macrophages of the innate immune system—educated in the tumor micro-environment—have emerged as prominent supporters of cancer progression that promote nine out of ten hallmarks of cancer. However, thanks to their remarkable intrinsic plasticity, macrophages retain the capability to promote the antitumor immune response even in thrall of cancer. Consequently, significant interest has been directed to the possibility of targeting tumor-associated macrophages to promote antitumor immunity as cancer immunotherapy. In this PhD thesis, I present the preclinical proof-of-concept, putative mechanism-of-action and results from early clinical trials of one such experimental immunotherapy: antibody blockade of the scavenger receptor Clever 1, expressed on a subset of tumor-associated macrophages. The results herein establish macro¬phage Clever 1 as an endogenous immune suppressor that restrains both macro¬phage overactivation and adaptive immunity. We show that Clever-1 blockade “re-edu¬cates” macrophages to promote antitumor immunity by activating cytotoxic T cells in preclinical tumor models. We propose this is mechanistically linked to Clever 1’s association with the vacuolar ATPase, the disruption of which antagonizes antigen degradation in phagolysosomes and saves them for cross-presenta¬tion. Lastly, I present results from early clinical trials, which indicate that Clever-1 blockade may boost antitumor immunity specifically in a subset of patients with noninflamed tumors for whom checkpoint blockade is rarely efficacious.-- Immuunijärjestelmä on herkeämätön suojamme sekä ulkoisia uhkatekijöitä että omia orastavia pahanlaatuisia solujamme vastaan. Valitettavasti kehittyvät syövät oppivat usein sammuttamaan kasvainta torjuvan immuunivasteen tai jopa keplottelevat immuunijärjestelmän edistämään niiden omaa kasvua ja etenemistä. Tarkastuspisteiden estäjät, jotka käynnistävät uudelleen kasvainta torjuvan hankitun immuniteetin, ovat mullistaneet syövän hoidon — niistä hyötyvät ovat kuitenkin vähemmistössä. Siksi tarvitaan uusia hoitovaihtoehtoja potilaille, joiden syövät ovat itsepintaisia. Synnynnäiseen immuniteettiin kuuluvat kasvaimeen liittyvät syöjäsolut — jotka koulutetaan kasvaimen mikroympäristössä — ovat syövän etenemisen keskeisiä tukijoita ja edistävät yhdeksää kymmenestä syövän ominaispiirteestä. Omaleimaisen luontaisen mukautumiskykynsä ansiosta syöjäsolut kuitenkin säilyttävät kykynsä myös vahvistaa kasvainta torjuvaa immuunivastetta jopa syövän pauloissa. Mahdollisuuteen herättää kasvainta torjuva immuniteetti käyttämällä kasvaimeen liittyviä syöjäsoluja lääkekohteena syövän immunologisessa hoidossa onkin kohdistunut merkittävää kiinnostusta. Tässä väitöskirjassa esitän prekliinisen soveltuvuusselvityksen, oletetun vaikutusmekanismin sekä tuloksia varhaisista kliinisistä kokeista yhdelle tällaiselle kokeelliselle immunoterapialle: vasta-ainevälitteiselle Clever-1-haaskareseptorin estolle, jota ilmentää osajoukko kasvaimeen liittyviä syöjäsoluja. Nämä tulokset vakiinnuttavat Clever-1:n sisäsyntyisenä immuniteetin vaimentajana, joka hillitsee sekä syöjäsolujen tarmokkuutta että hankittua immuniteettia. Osoitamme, että Clever-1:n häiritseminen ”uudelleenkouluttaa” syöjäsolut tukemaan kasvainta torjuvaa immuniteettia herättämällä tappaja-T-soluja prekliinisissä kasvainmalleissa. Mekaanisesti ehdotamme tämän olevan seurausta Clever-1:n ja vakuolaarisen ATPaasin yhteistoiminnasta, jonka purkaminen estää antigeenien pilkkomista lysosomeissa ja pelastaa ne ristiinesittelyä varten. Viimeiseksi esitän tuloksia varhaisista kliinisistä kokeista, joiden perusteella Clever-1:n estäminen saattaa tehostaa kasvainta torjuvaa immuniteettia erityisesti osajoukossa potilaita, joiden kasvaimissa ei ole aktiivista tulehdusvastetta ja joihin tarkastuspisteiden estäjät harvoin tehoavat

High-throughput cell-based compound screen identifies pinosylvin methyl ether and tanshinone IIA as inhibitors of castration-resistant prostate cancer

Current treatment options for castration-resistant prostate cancer (CRPC) are limited. In this study, a high-throughput screen of 4910 drugs and drug-like molecules was performed to identify antiproliferative compounds in androgen ablated prostate cancer cells. The effect of compounds on cell viability was compared in androgen ablated LNCaP prostate cancer cells and in LNCaP cells grown in presence of androgens as well as in two non-malignant prostate epithelial cells (RWPE-1 and EP156T). Validation experiments of cancer specific anti-proliferative compounds indicated pinosylvin methyl ether (PSME) and tanshinone IIA as potent inhibitors of androgen ablated LNCaP cell proliferation. PSME is a stilbene compound with no previously described antineoplastic activity whereas tanshinone IIA is currently used in cardiovascular disorders and proposed as a cancer drug. To gain insights into growth inhibitory mechanisms in CRPC, genome-wide gene expression analysis was performed in PSME- and tanshinone IIA-exposed cells. Both compounds altered the expression of genes involved in cell cycle and steroid and cholesterol biosynthesis in androgen ablated LNCaP cells. Decrease in androgen signalling was confirmed by reduced expression of androgen receptor and prostate specific antigen in PSME- or tanshinone IIA-exposed cells. Taken together, this systematic screen identified a novel anti-proliferative agent, PSME, for CRPC. Moreover, our screen confirmed tanshinone IIA as well as several other compounds as potential prostate cancer growth inhibitors also in androgen ablated prostate cancer cells. These results provide valuable starting points for preclinical and clinical studies for CRPC treatment

Enhanced Antibody Production in Clever-1/Stabilin-1–Deficient Mice

Clever-1, encoded by the Stab1 gene, is a scavenger and leukocyte trafficking receptor expressed by subsets of vascular and lymphatic endothelial cells and immunosuppressive macrophages. Monocyte Clever-1 also modulates T cell activation. However, nothing is known about the possible links between B cell function and Clever-1. Here, we found that Stab1 knockout mice (Stab1−/−) lacking the Clever-1 protein from all cells present with abnormally high antibody levels under resting conditions and show enhanced humoral immune responses after immunization with protein and carbohydrate antigens. Removal of the spleen does not abolish the augmented basal and post-immunization antibody levels in Clever-1–deficient mice. The increased IgG production is also present in mice in which Clever-1 is selectively ablated from macrophages. When compared to wildtype macrophages, Clever-1–deficient macrophages show increased TNF-α synthesis. In co-culture experiments, monocytes/macrophages deficient of Clever-1 support higher IgM production by B cells, which is blocked by TNF-α depletion. Collectively, our data show that the excessive inflammatory activity of monocytes/macrophages in the absence of Clever-1 results in augmented humoral immune responses in vivo

The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

Peer reviewe

Systemic Blockade of Clever-1 Elicits Lymphocyte Activation Alongside Checkpoint Molecule Downregulation in Patients with Solid Tumors : Results from a Phase I/II Clinical Trial

Purpose: Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell-targeting therapies. Thus, agents able to reprogram macrophages toward a proinflammatory state hold promise as novel immunotherapies for solid cancers. Inhibition of the macrophage scavenger receptor Clever-1 has shown benefit in inducing CD8 T-cell-mediated antitumor responses in mouse models of cancer, which supports the clinical development of Clever-1-targeting antibodies for cancer treatment. Patients and Methods: In this study, we analyzed the mode of action of a humanized IgG4 anti-Clever-1 antibody, FP-1305 (bexmarilimab), both in vitro and in patients with heavily pretreated metastatic cancer (n = 30) participating in part 1 (dose-finding) of a phase I/II open-label trial (NCT03733990). We studied the Clever-1 interactome in primary human macrophages in antibody pull-down assays and utilized mass cytometry, RNA sequencing, and cytokine profiling to evaluate FP-1305-induced systemic immune activation in patients with cancer. Results: Our pull-down assays and functional studies indicated that FP-1305 impaired multiprotein vacuolar ATPase-mediated endosomal acidification and improved the ability of macrophages to activate CD8(+)T-cells. In patients with cancer, FP-1305 administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. These effects were accompanied by a significant increase and activation of peripheral T-cells with indications of antitumor responses in some patients. Conclusions: Our results reveal a nonredundant role played by the receptor Clever-1 in suppressing adaptive immune cells in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch, potentially leading to intratumoral proinflammatory responses in patients with metastatic cancer.Peer reviewe

Systemic Blockade of Clever-1 Elicits Lymphocyte Activation Alongside Checkpoint Molecule Downregulation in Patients with Solid Tumors: Results from a Phase I/II Clinical Trial

Purpose:Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell–targeting therapies. Thus, agents able to reprogram macrophages toward a proinflammatory state hold promise as novel immunotherapies for solid cancers. Inhibition of the macrophage scavenger receptor Clever-1 has shown benefit in inducing CD8+ T-cell–mediated antitumor responses in mouse models of cancer, which supports the clinical development of Clever-1–targeting antibodies for cancer treatment.Patients and Methods:In this study, we analyzed the mode of action of a humanized IgG4 anti–Clever-1 antibody, FP-1305 (bexmarilimab), both in vitro and in patients with heavily pretreated metastatic cancer (n = 30) participating in part 1 (dose-finding) of a phase I/II open-label trial (NCT03733990). We studied the Clever-1 interactome in primary human macrophages in antibody pull-down assays and utilized mass cytometry, RNA sequencing, and cytokine profiling to evaluate FP-1305–induced systemic immune activation in patients with cancer.Results:Our pull-down assays and functional studies indicated that FP-1305 impaired multiprotein vacuolar ATPase–mediated endosomal acidification and improved the ability of macrophages to activate CD8+ T-cells. In patients with cancer, FP-1305 administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. These effects were accompanied by a significant increase and activation of peripheral T-cells with indications of antitumor responses in some patients.Conclusions:Our results reveal a nonredundant role played by the receptor Clever-1 in suppressing adaptive immune cells in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch, potentially leading to intratumoral</p

Crystal structure of ethyl (6-hydroxy-1-benzofuran-3-yl)acetate sesquihydrate

In the title hydrate, C12H12O4·1.5H2O, one of the water molecules in the asymmetric unit is located on a twofold rotation axis. The molecule of the benzofuran derivative is essentially planar (r.m.s. deviation for the non-H atoms = 0.021à ), with the ester group adopting a fully extended conformation. In the crystal, O-H�O hydrogen bonds between the water molecules and the hydroxy groups generate a centrosymmetric R6 6(12) ring motif. These R6 6(12) rings are fused, forming a one-dimensional motif extending along the c-axis direction

Disruption of Clever-1 for Novel Anticancer Immunotherapy

The progression of cancer is dependent on the quality of the immune response elicited by the tumour. Typically, proinflammatory immune responses restrain and anti-inflammatory immune responses promote the progression of cancer. The immunosuppression caused by anti-inflammatory leukocytes, such as regulatory T cells and alternatively activated macrophages, is a major obstacle in the successful treatment of cancer. Clever-1 is a multifunctional protein expressed by a subset of human monocytes and alternatively activated, or M2, macrophages. In these macrophages, Clever-1 is involved in receptor-mediated endocytosis, intracellular sorting, and transcytosis. Clever-1 is also expressed on lymphatic and vascular endothelia, where it mediates the trafficking of leukocytes from the lymph or blood into tissues. Lymphatics and macrophages expressing Clever-1 have been detected in human cancers, and high numbers of Clever-1-positive macrophages correlate with poorer prognoses in advanced colorectal cancer. By using specific antibodies to interfere with the function Clever-1, we have recently demonstrated that Clever-1 antibody treatment limits the progression of cancer in animal models of melanoma and lymphoma. According to these results, the antibody-mediated interference of Clever-1 both before and after cancer cell implantation limits tumour growth and metastasis. However, the molecular mechanisms by which Clever-1 regulates the phenotype and function of tumour-associated macrophages during this process are very poorly understood. The data presented in this master’s thesis reveal novel molecular mechanisms employed by Clever-1 to regulate the function of M2 macrophages and suggest that Clever-1 antibody treatment may have immunotherapeutic potential in the treatment of breast cancer. According to the obtained results, Clever-1 promoted intracellular signal transduction through the anti-inflammatory mTORC1 complex in M2 macrophages. In accordance, the antibody-mediated interference of Clever-1 induced a proinflammatory response from M2 macrophages by increasing the production and secretion of TNF-α, a major mediator of inflammation. Furthermore, combinatorial antibody-mediated interference of Clever-1 and the immune checkpoint protein PD-1 decreased the relative amount of anti-inflammatory tumour-associated macrophages, thereby likely promoting a more proinflammatory milieu within the tumour microenvironment. Additionally, combinatorial Clever-1 and PD-1 antibody treatment appeared to limit tumour growth and metastasis also in a mouse model of triple-negative breast cancer.Syövän eteneminen riippuu siitä, minkälaisen immuunivasteen kasvain aiheuttaa. Tavallisesti tulehdusta lisäävä eli proinflammatorinen immuunivaste estää ja tulehdusta vähentävä eli anti-inflammatorinen immuunivaste edistää syövän etenemistä. Anti-inflammatoristen valkosolujen, kuten säätelijä- T-solujen ja M2-makrofagien, aiheuttama immuunivasteen vaimennus on yksi onnistuneen syöpähoidon suurimmista esteistä. Clever-1 on monitoiminen proteiini, jota ilmentää ihmisen monosyyttien ja M2-makrofagien alaluokka. Näissä makrofageissa Clever-1 osallistuu reseptorivälitteiseen endosytoosiin, solunsisäiseen lajitteluun ja transsytoosiin. Clever-1:tä ilmennetään myös imu- ja verisuonten endoteelissä, missä se välittää valkosolujen kulkeutumista imunesteestä ja verestä kudoksiin. Ihmisen syövissä on havaittu Clever-1:tä ilmentäviä imusuonia ja makrofageja, ja esimerkiksi edenneessä peräsuolen syövässä suuri määrä Clever-1-positiivisia makrofageja korreloi heikomman ennusteen kanssa. Häiritsemällä Clever-1:n toimintaa siihen kohdennetuilla vasta-aineilla ryhmämme on hiljattain osoittanut, että Clever-1-vasta-ainehoito hidastaa syövän etenemistä melanooman ja lymfooman eläinmalleissa. Tulosten perusteella Clever-1:n toiminnan häiritseminen vasta-aineella sekä ennen syöpäsolujen istutusta että sen jälkeen vähentää kasvaimen kasvua ja etäpesäkkeiden muodostumista. Mekanismi, jolla Clever-1 säätelee kasvaimeen liittyvien makrofagien fenotyyppiä ja toimintaa kasvaimen kasvun ja etäpesäkkeiden muodostumisen aikana, on kuitenkin vielä erittäin puutteellisesti tunnettu. Tässä pro gradu -työssä esitetyt tulokset paljastavat uusia molekulaarisia mekanismeja, joilla Clever-1 säätelee M2-makrofagien toimintaa, ja antavat viitteitä Clever-1-vasta-ainehoidon mahdollisesta immunoterapeuttisesta tehosta myös rintasyövän hoidossa. Saatujen tulosten perusteella Clever-1 lisäsi anti-inflammatorisen mTORC1-kompleksin kautta tapahtuvaa solunsisäistä signaalitransduktiota M2-makrofageissa. Vastaavasti Clever-1:n toiminnan häiritseminen vasta-aineella aiheutti M2-makrofageissa proinflammatorisen vasteen lisäämällä keskeisen tulehduksenvälittäjäaineen tuumorinekroositekijä- α:n tuotantoa ja erittymistä. Lisäksi yhdistelmähoito Clever-1-:tä ja PD-1-säätelijäproteiinia estävällä vasta-aineella vähensi kasvaimeen liittyvien anti-inflammatoristen makrofagien suhteellista osuutta ja siten todennäköisesti edisti proinflammatorista tulehdusvastetta kasvaimen sisällä. Sen lisäksi yhdistelmähoito Clever-1- ja PD-1-vasta-aineella näytti estävän kasvaimen kasvua ja etäpesäkkeiden muodostumista triplanegatiivisen rintasyövän hiirimallissa.Siirretty Doriast

Cryptocurrencies as a future payment method

Tutkimuksen tavoitteena on tarkastella kryptovaluuttoja niin maksutapana sekä mahdollisuutta tulla yleisesti käytetyksi valuutaksi, kuten euro ja dollari. Tutkimus hakee vastausta kysymykseen ’’Voiko kryptovaluutoista tulla laajalti käytetty maksutapa?’’ Työ on kartoittava sekä ennustava tutkimus, joka kartoittaa kirjallisuutta ja sen sisältöä tietyllä aihealueella sekä pyrkii arvioimaan kryptovaluuttojen ilmenemismuotoja tulevaisuudessa. Teoriaosuuden materiaali on kerätty kirjallisuudesta sekä internet-lähteistä. Teoria koostuu viidestä osuudesta kryptovaluutat, lohkoketju, lompakot, maksutavat ja säännökset. Bitcoin oli ensimmäinen kryptovaluutta, jonka jälkeen on tullut tuhansia muita. Kaikkien kryptovaluuttojen yhteenlaskettu arvo on ylittänyt 2 biljoonaa dollaria. Tutkimuksen tuloksista saadaan selville, että kryptovaluutoista voi tulla laajasti käytetty maksuväline tulevaisuudessa. Tämä vaatii kuitenkin valtioilta tarkkaa sääntelyä niihin liittyen, jotta kryptovaluuttojen tarjoajilla sekä käyttäjillä on selvät säännöt. Kryptovaluutat ovat yleistyneet maailmalla, mutta niillä on vielä haasteita ylitettävänä yleisesti käytetyksi maksutavaksi.The study aimed to look at cryptocurrencies as a method of payment and the possibility of cryptocurrencies to become a commonly used currency, such as the euro and the dollar are. The study sought an answer to the question, ‘’Can cryptocurrencies become a widely used payment method?’’ The work is a mapping and predictive study that maps the literature and its content in a specific topic area and seeks to evaluate the manifestations of cryptocurrencies in the future. The material of the theoretical section was collected from the literature and internet sources. The theory consists of five topic areas: cryptocurrencies, blockchain, cryptocurrency wallets, payment methods, and regulations. Bitcoin was the first cryptocurrency and was followed by thousands of others. The total market cap of all cryptocurrencies is 2 trillion dollars. The study results showed that cryptocurrencies may become a widely used means of payment in the future. However, it will require precise regulation from the countries to have clear rules for cryptocurrency providers and users. Cryptocurrencies have become more widespread worldwide, but they still have challenges to overcome before becoming a commonly used payment method